Letter | Published:

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

Nature volume 553, pages 8690 (04 January 2018) | Download Citation

Abstract

The mammalian microbiome has many important roles in health and disease1,2, and genetic engineering is enabling the development of microbial therapeutics and diagnostics3,4,5,6,7. A key determinant of the activity of both natural and engineered microorganisms in vivo is their location within the host organism8,9. However, existing methods for imaging cellular location and function, primarily based on optical reporter genes, have limited deep tissue performance owing to light scattering or require radioactive tracers10,11,12. Here we introduce acoustic reporter genes, which are genetic constructs that allow bacterial gene expression to be visualized in vivo using ultrasound, a widely available inexpensive technique with deep tissue penetration and high spatial resolution13,14,15. These constructs are based on gas vesicles, a unique class of gas-filled protein nanostructures that are expressed primarily in water-dwelling photosynthetic organisms as a means to regulate buoyancy16,17. Heterologous expression of engineered gene clusters encoding gas vesicles allows Escherichia coli and Salmonella typhimurium to be imaged noninvasively at volumetric densities below 0.01% with a resolution of less than 100 μm. We demonstrate the imaging of engineered cells in vivo in proof-of-concept models of gastrointestinal and tumour localization, and develop acoustically distinct reporters that enable multiplexed imaging of cellular populations. This technology equips microbial cells with a means to be visualized deep inside mammalian hosts, facilitating the study of the mammalian microbiome and the development of diagnostic and therapeutic cellular agents.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & The gut microbiota shapes intestinal immune responses during health and disease. Nat. Rev. Immunol. 9, 313–323 (2009)

  2. 2.

    & The role of microbiome in central nervous system disorders. Brain Behav. Immun. 38, 1–12 (2014)

  3. 3.

    et al. Programmable probiotics for detection of cancer in urine. Sci. Transl. Med. 7, 289ra284 (2015)

  4. 4.

    et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289, 1352–1355 (2000)

  5. 5.

    & Synthetic microbes as drug delivery systems. ACS Synth. Biol. 4, 358–364 (2015)

  6. 6.

    et al. Synchronized cycles of bacterial lysis for in vivo delivery. Nature 536, 81–85 (2016)

  7. 7.

    et al. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation. Nat. Biotechnol. 35, 653–658 (2017)

  8. 8.

    , & Gut biogeography of the bacterial microbiota. Nat. Rev. Microbiol. 14, 20–32 (2016)

  9. 9.

    & . Fate, activity, and impact of ingested bacteria within the human gut microbiota. Trends Microbiol. 23, 354–366 (2015)

  10. 10.

    , , , & In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice. Appl. Environ. Microbiol. 76, 264–274 (2010)

  11. 11.

    , , , & Bioluminescence imaging study of spatial and temporal persistence of Lactobacillus plantarum and Lactococcus lactis in living mice. Appl. Environ. Microbiol. 79, 1086–1094 (2013)

  12. 12.

    et al. A bright cyan–excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nat. Biotechnol. 34, 760–767 (2016)

  13. 13.

    et al. Use of diagnostic imaging studies and associated radiation exposure for patients enrolled in large integrated health care systems, 1996-2010. J. Am. Med. Assoc. 307, 2400–2409 (2012)

  14. 14.

    et al. Principles and applications of ultrasound backscatter microscopy. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 40, 608–617 (1993)

  15. 15.

    et al. Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging. Nature 527, 499–502 (2015)

  16. 16.

    Gas vesicles. Microbiol. Rev. 58, 94–144 (1994)

  17. 17.

    Distribution, formation and regulation of gas vesicles. Nat. Rev. Microbiol. 10, 705–715 (2012)

  18. 18.

    et al. Biogenic gas nanostructures as ultrasonic molecular reporters. Nat. Nanotechnol. 9, 311–316 (2014)

  19. 19.

    & Gas vesicle genes identified in Bacillus megaterium and functional expression in Escherichia coli. J. Bacteriol. 180, 2450–2458 (1998)

  20. 20.

    et al. Molecular engineering of acoustic protein nanostructures. ACS Nano 10, 7314–7322 (2016)

  21. 21.

    in Medical Microbiology 4th edn (ed. ) Ch. 95 (Univ. Texas Medical Branch, 1996)

  22. 22.

    & Bacterial growth: global effects on gene expression, growth feedback and proteome partition. Curr. Opin. Biotechnol. 28, 96–102 (2014)

  23. 23.

    , & Gas vesicles are strengthened by the outer-surface protein, GvpC. Arch. Microbiol. 157, 229–234 (1992)

  24. 24.

    , , & Recombinant lactic acid bacteria as mucosal biotherapeutic agents. Trends Biotechnol. 29, 499–508 (2011)

  25. 25.

    & The non-pathogenic Escherichia coli strain Nissle 1917—features of a versatile probiotic. Microb. Ecol. Health Dis. 21, 122–158 (2009)

  26. 26.

    et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J. Clin. Invest. 124, 3391–3406 (2014)

  27. 27.

    et al. Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct. Infect. Immun. 68, 3594–3600 (2000)

  28. 28.

    , , & Overloaded and stressed: whole-cell considerations for bacterial synthetic biology. Curr. Opin. Microbiol. 33, 123–130 (2016)

  29. 29.

    & Visualization of evolutionary stability dynamics and competitive fitness of Escherichia coli engineered with randomized multigene circuits. ACS Synth. Biol. 2, 519–528 (2013)

  30. 30.

    , , , & In vivo gene expression dynamics of tumor-targeted bacteria. ACS Synth. Biol. 1, 465–470 (2012)

  31. 31.

    & Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009)

  32. 32.

    et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004)

  33. 33.

    et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013)

  34. 34.

    et al. Development of strain-specific PCR reactions for the detection of the probiotic Escherichia coli strain Nissle 1917 in fecal samples. Res. Microbiol. 154, 59–66 (2003)

  35. 35.

    et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012)

  36. 36.

    et al. Molecular imaging of inflammation in inflammatory bowel disease with a clinically translatable dual-selectin-targeted US contrast agent: comparison with FDG PET/CT in a mouse model. Radiology 267, 818–829 (2013)

  37. 37.

    & Assessment of murine colorectal cancer by micro-ultrasound using three dimensional reconstruction and non-linear contrast imaging. Mol. Ther. Methods Clin. Dev. 3, 16070 (2016)

Download references

Acknowledgements

We thank F. S. Foster, D. Maresca, A. Mukherjee, M. Din, T. Danino, J. Willmann and S. K. Mazmanian for discussions, and A. McDowall for assistance with electron microscopy. This research was supported by the National Institutes of Health grant R01-EB018975, the Canadian Institute of Health Research grant MOP 136842 and the Pew Scholarship in the Biomedical Sciences. A.L. is supported by the NSF graduate research fellowship (award 1144469) and the Biotechnology Leaders Program. A.F. is supported by the NSERC graduate fellowship. S.P.N. was supported by the Caltech Summer Undergraduate Research Fellowship. Research in the Shapiro laboratory is also supported by the Heritage Medical Research Institute, the Burroughs Wellcome Career Award at the Scientific Interface and the David and Lucile Packard Fellowship for Science and Engineering.

Author information

Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA

    • Raymond W. Bourdeau
    • , Audrey Lee-Gosselin
    • , Suchita P. Nety
    •  & Mikhail G. Shapiro
  2. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA

    • Anupama Lakshmanan
    • , Arash Farhadi
    •  & Sripriya Ravindra Kumar

Authors

  1. Search for Raymond W. Bourdeau in:

  2. Search for Audrey Lee-Gosselin in:

  3. Search for Anupama Lakshmanan in:

  4. Search for Arash Farhadi in:

  5. Search for Sripriya Ravindra Kumar in:

  6. Search for Suchita P. Nety in:

  7. Search for Mikhail G. Shapiro in:

Contributions

R.W.B. and M.G.S. conceived and designed the study. R.W.B., A.L., A.L.-G., A.F. and S.P.N. prepared genetic constructs in E. coli. R.W.B., A.L., A.L.-G., S.P.N. and A.F. conducted in vitro ultrasound experiments. A.L.-G. and R.W.B. performed in vivo ultrasound experiments. A.L., A.F. and A.L.-G. conducted metabolic burden experiments in Nissle 1917 cells. R.W.B. and S.R.K. prepared genetic constructs in S. typhimurium. R.W.B. and A.L. obtained TEM images. R.W.B., A.L.-G. and M.G.S. analysed ultrasound data. R.W.B. and M.G.S. wrote the manuscript with input from all authors. M.G.S. supervised the research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mikhail G. Shapiro.

Reviewer Information Nature thanks C. Caskey, O. Couture, P. Silver and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Notes 1–5, Plasmid Sequences for ARG1 and ARG2 constructs, Supplementary References and Supplementary Table 1.

  2. 2.

    Life Sciences reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature25021

Further reading Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.